Sirna conjugate and preparation method thereof

ABSTRACT

Provided are an siRNA-polymer conjugate, and a method for preparing the same, and more specifically, to a hybrid conjugate formed by covalently bonding siRNA and a polymeric compound for improving the in vivo stability of siRNA, and to a preparation method of the hybrid conjugate. The conjugate of the present invention can improve the in vivo stability of siRNA, thereby achieving an efficient delivery of therapeutic siRNA into cells and exhibiting the activity of siRNA even with a small dose of a relative low concentration. Therefore, the conjugate can advantageously be used as not only an siRNA treatment tool for cancers and other infectious disease, but also a novel type siRNA delivery system.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application under the provisions of 35U.S.C. §120 of U.S. patent application Ser. No. 13/319,885 filed Nov.10, 2011, which is a U.S. national phase application under theprovisions of 35 U.S.C. §371 of International Patent Application No.PCT/KR10/03039 filed May 13, 2010, which in turn claims priority ofKorean Patent Application No. 10-2009-0042297 filed May 14, 2009. Thedisclosures of such U.S. and international patent applications andKorean priority patent application are hereby incorporated herein byreference in their respective entireties, for all purposes.

TECHNICAL FIELD

The present invention relates to a conjugate in which a polymer compoundfor improving delivery of siRNA useful in gene therapy of cancers andother infectious diseases is conjugated to the siRNA by using adegradable or a non-degradable bond, a method for preparing theconjugate, and a method for delivering the siRNA using the conjugate.

BACKGROUND ART

RNA interference refers to a mechanism, which is post-transcriptionalgene silencing initiated by a double-stranded RNA (dsRNA) via nucleotidesequence specific manner in a gene expression process, and thismechanism is first found in C. elegans, and commonly found in plant,fruitfly, and vertebrate (Fire et al., Nature, 391:806-811, 1998; Novina& Sharp, Nature, 430:161-164, 2004). It has been known that RNAinterference occurs in such a manner that dsRNA of 19-25 bp entering inthe cell is bound with an RISC (RNA-induced silencing complex), and onlyan antisense (guide) strand is bound with mRNA such that it iscomplementary to the nucelotide sequence of the mRNA, thereby degradingtarget mRNA by endonuclease domains existing in the RICS (Rana, T. M.,Nat. Rev. Mol. Cell Biol., 8:23-36, 2007; Tomari, Y. and Zamore, P. D.,Genes Dev., 19: 517-529, 2005).

When the dsRNA is delivered into a cell, it is specifically bound to atarget mRNA sequence to degrade the mRNA, and thereby, it is consideredas a new tool capable of regulating gene expression. However, in case ofhuman, it was difficult to obtain RNAi effect due to the induction of anantiviral interferon pathway on introduction of dsRNA into human cells.In 2001, Elbashir and Tuschl et al., found that the introduction ofsmall dsRNA of 21nt length (nucleotides length) into human cells did notcause the interferon pathway but specifically degraded the target mRNA(Elbashir, S. M., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K.,Tuschl, T., Nature, 411, 494-498, 2001; Elbashir, S. M., Lendeckel, W.,Tuschl, T., Genes & Dev., 15, 188-200, 2001; Elbashir, S. M., Martinez,J., Patkaniowska, A., Lendeckel, W., Tuschl, T., EMBO J., 20, 6877-6888,2001). Thereafter, dsRNA of 21nt length has been spotlighted as a toolof new functional genomics and named as small interfering RNA (siRNA).

The siRNA is a substance gaining a lot of interest as a agent for genetherapy ever since it was reported to have an excellent effect ininhibiting expression of a specific gene in animal cells. In effect,because of its high activity and precise gene selectivity, siRNA isexpected to be an alternative therapeutic agent to an antisenseoligonucleotide (ODN) currently being used as a therapeutic agent, as aresult of a 20-year research (Dana J. Gary et al. Journal of ControlledRelease 121:64-73, 2007). A siRNA technique aiming to therapy has greatadvantages in that it is easily designed compared with other medicinesand has high target selectivity and a property of inhibiting expressionof a specific gene. In addition, it is less toxic because RNAinterference suppresses gene expression by using a mechanism naturallyexisting in a living system. ‘Bevasiranib’, recently developed as atherapeutic agent for wet age-related macular disease by OPKO Inc., is asiRNA which acts selectively on a vascular endotherial growth factor(VEGF) inducing neovascularization to inhibit expression of the VEGF,and passes through three phases of clinical trial (Dejneka N S et al.,Mol Vis., 28(14):997-1005, 2008). Besides, therapeutic agents includingsiRNAs targeting various genes are currently being developed (Ryan P.Million, Nature Reviews Drug Discovery 7: 115-116, 2008).

Despite various results showing that specific expression inhibition isinduced in vivo through RNA interference, in vivo siRNA delivery hasmany problems to be solved, such as degradation by enzymes in the blood,interaction with components in the blood, and non-specific delivery tocells (Shigeru Kawakami and Mitsuru Hashida, Drug Metab. Pharmacokinet.22(3): 142-151, 2007). Attempts to overcome these problems are inprogress by partially using nuclease resistant nucleoside analogues orimproving delivery techniques.

Examples of the improved delivery techniques include gene deliverytechniques using viruses such as adenoviruses, retroviruses, etc., andgene delivery techniques by non-viral vectors using liposomes, cationiclipid, and cationic polymer compounds. However, viral carriers has aproblem in safety since delivered genes are likely to be integrated intoa chromosome of a host to induce abnormality in normal functions ofgenes of the host and activate oncogenes, and in addition, may causeautoimmune diseases due to successive expression of viral genes even insmall amounts, or may not lead to efficient protective immunity in acase where modified viral infection is induced from the viral carriers.Meanwhile, non-viral carriers are less efficient than the viralcarriers, but have advantages of low side effects and inexpensiveproduction costs, considering in vivo safety and economic feasibility(Lehrman S., Nature. 401(6753): 517-518, 1999). In addition, non-viraldelivery methods require to effectively protect enzymatic ornon-enzymatic degradation in order to deliver RNA molecules includingsiRNA, one method of which is to utilize DNA expression plasmidsencoding a short hairpin RNA (shRNA). A system through DNA has anadvantage in that siRNA is expressed only while an expression vectorexists. Moreover, a recent study on chemical modification of siRNA hasproposed a method for improving the stability against nucleases and thelow intracellular uptake (Shigery Kawakami and Mitsuru Hashida. DrugMetab. Parmacokinet. 22(3): 142-151, 2007).

In one type of chemical modification of siRNA, a phosphorodiester bond,which is a part degraded by the nuclease, was modified with aphosphorothioate linkage or the 2′ portion of a pentose is modified with2′-O-meRNA, 2′-deoxy-2′-fluouridine, or a locked nucleic acid (LNA)formed by linking the 2′ portion and the 4′ portion, and as a result,the stability in the serum was improved ((Braasch D. A. et al. Bioorg.Med. Chem. Lett. 14:1139-1143, 2003; Chiu Y. L. and Rana T. M., RNA,9:1034-1048, 2003; Amarzguioui M. et al. Nucleic Acid Res. 31:589-595,2003). In another type of chemical modification, a functional group islinked to a 3′-end region of a sense (anti-guide) strand, resulting inimprovement in pharmacokinetic characteristics compared with a control,and high efficiency is induced at the time of application in vivothrough a balance between hydrophilicity and hydrophobilicity of siRNA(Soutschek J. et al. Nature 432:173-178 2004).

However, the above methods still leave much to be desired in order toprotect siRNA from nucleases and improve the efficiency of cell-membranepermeability.

For that reason, the inventors have found that a conjugate, in whichhydrophilic or hydrophobic polymer compound is conjugated to siRNA byusing a degradable or a non-degradable bond, improved in vivo stabilityof siRNA, and, based on this, has completed the present invention.

DISCLOSURE Technical Problem

An object of the present invention is to provide a conjugate in which ahydrophilic or hydrophobic polymer compound, which is a biocompatiblepolymer compound, is conjugated to an end of a sense strand or anantisense strand of siRNA by using a degradable or non-degradable bond,in order to improve the intracellular delivery efficiency of the siRNA.

Another object of the present invention is to provide a solid supportcontaining a polymer compound, especially, a polymer compound of whichstability is proved when applied to human body, for example,polyethylene glycol (PEG), and a method for efficiently preparing anoligonucleotide including RNA, DNA, RNA-DNA chimera, and analog thereof,in which PEG is bound to the 3′ end thereof by using the support.

Still another object of the present invention is to provide a method forpreparing the siRNA conjugate and a method for delivering siRNA usingthe siRNA conjugate.

Technical Solution

In order to achieve the above objects, a first of the present inventionprovides an siRNA-polymer compound conjugate of the following structure:

A-X-R-Y-B

(wherein, A and B are independently a hydrophilic polymer or hydrophobicpolymer compound; X and Y are independently a simple covalent bond or alinker-mediated covalent bond; and R is siRNA).

A second of the present invention provides an siRNA-polymer compoundconjugate of the following structure:

A-X-R

(wherein, A is a hydrophobic polymer compound; X is a simple covalentbond or a linker-mediated covalent bond; and R is siRNA).

A third of the present invention provides a conjugate in which a singlestrand of the siRNA (R) is composed of 19 to 31 nucleotides.

A fourth of the present invention provides a conjugate in which thehydrophobic polymer compound (A) has a molecular weight of 250 to 1,000.

A fifth of the present invention provides a conjugate in which thehydrophobic polymer compound (A) is C₁₆˜C₅₀ hydrocarbon or cholesterol.

A sixth of the present invention provides a conjugate in which thecovalent bond (X, Y) is a non-degradable bond or a degradable bond.

A seventh of the present invention provides a conjugate in which thenon-degradable bond is an amide bond or a phosphate bond.

An eighth of the present invention provides a conjugate in which thedegradable bond is selected from a disulfide bond, an acid-cleavablebond, an ester bond, an anhydride bond, a biodegradable bond and anenzyme-cleavable bond.

A ninth of the present invention provides a conjugate in which thehydrophilic polymer compound (A or B) is a non-ionic polymer compoundhaving a molecular weight of 1,000 to 10,000.

A tenth of the present invention provides a conjugate in which thehydrophilic polymer compound is selected from a group consisting ofpolyethylene glycol (PEG), polyvinylpyrolidone, and polyoxazoline.

An eleventh of the present invention provides a polyethyleneglycol-bound solid support of the following structure:

[where, R is alkyl, alkenyl, alkynyl, aryl, arylalkyl, heteroalkyl, orheteroaryl; m is an integer of 2 to 18; n is an integer of 5 to 120; andX is hydrogen, 4-monomethoxytrityl, 4,4′-dimethoxytrityl, or4,4′,4″-trimethoxytrityl].

A twelfth of the present invention provides a polyethylene glycol-boundsolid support in which the solid support is controlled pore glass (CPG).

A thirteenth of the present invention provides a polyethyleneglycol-bound solid support in which the CPG has a diameter of 40˜180 μmand a pore size of 500 Å˜3000 Å.

A fourteenth of the present invention provides a polyethyleneglycol-bound solid support which is 3′-PEG-CPG having the followingstructural formula IV:

A fifteenth of the present invention provides a method for preparing3′-PEG-CPG the method including:

1) reacting CPG with 3-aminopropyltriethoxysilane to form long chainalkyl amine controlled pore glass (LCAA-CPG);

2) reacting polyethylene glycol with 4,4′-dimethoxytrityl chloride toform 2-[bis-(4-dimethoxytrityl)-poly(ethylene glycol)];

3) reacting the compound formed in the step 2) and a compound of thefollowing chemical formula 1 to form a compound of the followingstructural formula I;

4) reacting the formed compound of the following structural formula Iand 4-nitrophenylchloroformate to form a compound of the followingstructural formula II;

5) reacting the compound of the following structural formula I formed inthe step 3) and N-succinimidyl trifluoroacetic acid to form a compoundof the following structural formula III; and

6) reacting the LCAA-CPG compound formed in the step 1) with thecompounds of the following structural formulas I, II, and IIIrespectively formed in the steps 3) to 5), respectively.

[where, R is alkyl, alkenyl, alkynyl, aryl, arylalkyl, heteroalkyl, orheteroaryl; and n is an integer of no less than 5 and no more than 120].

A sixteenth of the present invention provides a method for preparing ansiRNA conjugate, the method including:

1) preparing an siRNA for a target gene by using the polyethyleneglycol-bound solid support of the eleventh of the present invention; and

2) linking an end group of the siRNA and polyethylene glycol by acovalent bond.

A seventeenth of the present invention provides a nanoparticleconsisting of siRNA conjugates of the first or second of the presentinvention.

An eighteenth of the present invention provides a method for genetherapy, including:

1) preparing the nanoparticles of the seventeenth of the presentinvention; and

2) administering the nanoparticles into the body of an animal.

A nineteenth of the present invention provides a method for gene therapyin which the nanoparticles are administered into the body by oraladministration or intravenous injection.

A twentieth of the present invention provides a pharmaceuticalcomposition including a pharmaceutically effective amount of the siRNAconjugates of the first or second of the present invention.

A twenty-first of the present invention provides a pharmaceuticalcomposition including a pharmaceutically effective amount of thenanoparticles of the seventeenth of the present invention.

Hereinafter, the present invention will be described in detail.

The present invention provides an siRNA-polymer compound conjugate ofthe following structure:

A-X-R-Y-B.

Wherein, A and B are independently a hydrophilic polymer or hydrophobicpolymer compound; X and Y are independently a simple covalent bond or alinker-mediated covalent bond; and R is siRNA.

Moreover, the present invention provides an siRNA-polymer compoundconjugate of the following structure:

A-X-R.

Wherein, A is a hydrophobic polymer compound; X is a simple covalentbond or a linker-mediated covalent bond; and R is siRNA.

In the conjugate of the present invention, an oligonucleotide strand ofthe siRNA may include 19 to 31 nucleotides. Any siRNA derived from genesthat is used or is likely to be used for gene therapy or study may beemployed as the siRNA usable in the present invention.

The hydrophobic polymer compound may be a hydrophobic polymer compoundhaving a molecular weight of 250 to 1,000. Examples of the hydrophobicpolymer compound may include hydrocarbon, preferably, C16˜C50hydrocarbon, and cholesterol. Here, the hydrophobic polymer compound isnot limited to only the hydrocarbon and the cholesterol.

The hydrophobic polymer compound causes a hydrophobic interaction tofunction to form a micelle consisting of siRNA-hydrophobic polymercompound conjugates. Among the hydrophobic polymer compounds,especially, the saturated hydrocarbon has an advantage in that it can beeasily conjugated to the siRNA during manufacturing of the siRNA, andthus, it is very suitable for manufacturing conjugates of the presentinvention.

Also, the covalent bond (i.e., X, Y) may be any one of a non-degradableor a degradable bond. Here, there may be an amide bond or a phosphatebond in the non-degradable bond, and there may be a disulfide bond, anacid-cleavable bond, an ester bond, an anhydride bond, a biodegradablebond and an enzyme-cleavable bond in the degradable bond. However, thenon-degradable or the degradable bond is not limited thereto.

The linker mediating the bond covalently binds the hydrophilic polymer(or the hydrophobic polymer) and an end of a residue derived from thesiRNA, and is not particularly limited as long as it can provide adegradable bond in a certain environment, as necessary. Therefore, thelinker may include any compound that can be bound with the siRNA and/orthe hydrophilic polymer (or the hydrophobic polymer) to activate themduring the manufacturing procedure of the conjugate.

Also, the hydrophilic polymer compound may be a non-ionic polymercompound having a molecular weight of 1,000 to 10,000. For example, thehydrophilic polymer compound may include a non-ionic hydrophilic polymercompound of polyethylene glycol, polyvinylpyrolidone, polyoxazoline, andthe like, but is not limited thereto.

A functional group of the hydrophilic polymer compound may be replacedby another functional group, as necessary. Among the hydrophilic polymercompounds, particularly, PEG is very suitable for manufacturing theconjugates of the present invention since it has various molecularweights, has an end capable of introducing functional groups, hasexcellent biocompatibility, does not induce immune reactions, andincreases the water-solubility to improve gene delivery efficiency invivo.

Moreover, the present invention provides a polyethylene glycol-boundsolid support of the following structure:

Wherein, the solid support includes, for example, CPG, polystyrene,silica gel, cellulose paper, etc., but is not necessarily limitedthereto; R is alkyl, alkenyl, alkynyl, aryl, arylalkyl, heteroalkyl, orheteroaryl; m is an integer of 2 to 18; n is an integer of 5 to 120(molar mass 282˜5300); and X is 4-monomethoxytrityl,4,4′-dimethoxytrityl, or 4,4′,4″-trimethoxytrityl] and removed afteracid treatment to become hydrogen. In a case where the solid support isCPG, it may have a diameter of 40˜180 μm and a pore size of 500 Å˜3000Å.

Also, the present invention provides a polyethylene glycol-bound solidsupport in which 3′-PEG-CPG having the following structural formula IVis bound:

Moreover, the present invention provides a method for preparing3′-PEG-CPG of the following structural formula IV, the method including:

1) reacting CPG with 3-aminopropyltriethoxysilane to form LCAA-CPG;

2) reacting polyethylene glycol with 4,4′-dimethoxytrityl chloride toform 2-[bis-(4-dimethoxytrityl)-poly(ethylene glycol)];

3) reacting the compound formed in the step 2) and a compound of thefollowing formula 1 to form a compound of the following structuralformula I;

4) reacting the formed compound of the following structural formula Iand 4-nitrophenylchloroformate to form a compound of the followingstructural formula II;

5) reacting the compound of the following structural formula I formed inthe step 3) and N-succinimidyl trifluoroacetic acid to form a compoundof the following structural formula III; and

6) reacting the LCAA-CPG compound formed in the step 1) with thecompounds of the following structural formulas I, II, and IIIrespectively formed in the steps 3) to 5), respectively.

[where, R is alkyl, alkenyl, alkynyl, aryl, arylalkyl, heteroalkyl, orheteroaryl; and n is an integer of no less than 5 and no more than 120].

Also, the present invention provides a method for preparing an conjugatecomprising siRNA and PEG by using the polyethylene glycol-bound solidsupport. More specifically, a method for preparing an siRNA conjugate isprovided, the method including:

1) preparing an siRNA for a target gene by using the polyethyleneglycol-bound solid support; and

2) linking an end group of the siRNA and polyethylene glycol by acovalent bond. Through this, oligonucleotides including RNA, DNA,RNA-DNA chimera, and analog thereof can be efficiently prepared.

According to a preferred embodiment of the present invention, the siRNAcan be prepared by linking phosphordiester bonds building an RNAbackbone structure, using β-cyanoethyl phosphoramidite (Shina et al.Nucleic Acids Research, 12:4539-4557, 1984). For example, a series ofprocedures consisting of deblocking, coupling, oxidation and cappingwere repeatedly performed on a sold support on which nucleotide wasattached, by using an RNA synthesizer, to obtain the reactant containinga desired length of RNA. However, the present invention is not limitedthereto.

Also, the present invention provides a nanoparticle consisting of siRNAconjugates.

The siRNA-polymer compound conjugates of the present invention can forma nanoparticle structure by interaction therebetween, and thesiRNA-polymer compound conjugate and the nanoparticle consisting of thesiRNA-polymer compound conjugates thus obtained improve intracellulardelivery of siRNA and can be applicable for therapeutic treatment ofdisease models. The preparation of conjugates, and characteristics andintracellular delivery efficiency and effect of the nanoparticleconsisting of the conjugates will be in detail described in the examplesto be described later.

Also, the present invention provides a method for gene therapy using thenanoparticle.

More specifically, the method for gene therapy includes preparing thenanoparticles each consisting of the siRNA-polymer compound conjugatesand administering the nanoparticles into the body of an animal.

Also, the present invention provides a pharmaceutical compositionincluding a pharmaceutically effective amount of the nanoparticles eachconsisting of the siRNA conjugates.

The composition of the present invention can be prepared to include oneor more of pharmaceutically acceptable carriers in addition to theabove-described active components, for administration. Thepharmaceutically acceptable carrier needs to be compatible with theactive components of the present invention. The pharmaceuticallyacceptable carrier may be used by mixing with saline solution,sterilized water, Ringer's solution, buffered saline solution, dextrosesolution, maltodextrin solution, glycerol and ethanol, and one or morethereof, and as necessary, other common additives such as antioxidants,buffer solution, bacteriostatic agents, or the like, may be addedthereto. In addition, diluents, dispersants, surfactants, binders, andlubricants can be adjunctively added thereto to formulate formulationsfor injection such as aqueous solution, suspension, emulsion, or thelike. Furthermore, the composition of the present invention can bepreferably formulated according to specific diseases or components, byusing appropriate methods in the art or methods disclosed in Remington'spharmaceutical Science (Mack Publishing company, Easton Pa.).

The pharmaceutical composition of the present invention can bedetermined by those skilled in the art, based on syndromes and diseaseseverity of patients. Also, the pharmaceutical composition of thepresent invention can be formulated in various types such as powder,tablet, capsule, liquid, injectable, ointment, syrup, and the like, andcan be provided in single-dosage or multi-dosage container, for example,a sealed ample, a bottle, or the like.

The pharmaceutical composition of the present invention can be orally orparenterally administered. The administration route of thepharmaceutical composition according to the present invention mayinclude, but is not limited to, oral, intravenous, intramuscular,intramedullary, intrathecal, intracardiac, dermal, subcutaneous,intraperitoneal, enteral, sublingual, or topical administration.

For this clinical administration, the pharmaceutical composition of thepresent invention can be formulated in an appropriate formulation byusing the known arts. The dosage of the composition of the presentinvention has various ranges depending on weight, age, gender, healthstatus, diet, administration time and method, excretion rate, anddisease severity of patient, and can be easily determined by thoseskilled in the art.

Advantageous Effects

The nanoparticle consisting of siRNA-polymer compound conjugates of thepresent invention can improve in vivo stability of siRNA to efficientlydeliver a therapeutic siRNA into the cell, and can be very useful in abasic research for biotechnology and medical industry as a new type ofsiRNA delivery system, as well as a tool for siRNA treatment of cancersand other infective diseases since it can exhibit siRNA activity in arelatively low concentration of dosage even without transfectionreagents.

DESCRIPTION OF DRAWINGS

FIG. 1 shows a structural formula of 3′-PEG-CPG prepared;

FIG. 2 shows ¹H NMR data of the compound obtained in Example 1;

FIG. 3 shows ¹H NMR data of [Compound A], which is a 3′-PEG reagent forbinding with LCAA-CPG in Example 1;

FIG. 4 shows ¹H NMR data of [Compound B], which is a 3′-PEG reagent forbinding with LCAA-CPG in Example 1;

FIG. 5 shows ¹H NMR data of [Compound C], which is a 3′-PEG reagent forbinding with LCAA-CPG in Example;

FIG. 6 shows Maldi-Tof molecular weight data after manufacturing of3′-PEG-CPG and an oligonucleotide (siRNA) in Example 1-3;

FIG. 7 shows Maldi-Tof molecular weight data after manufacturing of3′-PEG-CPG and an oligonucleotide (siRNA) in Example 1-4;

FIG. 8 shows an electrophoresis photograph of a naked siRNA in whichnone of polymer compounds are conjugated, and siRNA-polymer compoundconjugates in which a hydrophilic or hydrophobic polymer compound isconjugated (The siRNA means naked siRNA and respective conjugatesrepresent siRNA-polymer compound conjugates shown in Table 1. Also,19mer, 23mer, 27mer, and 31mer mean siRNAs consisting of 19, 23, 27, and31 nucleotides, respectively, and they all were used to preparesiRNA-polymer compound conjugates in a structure of the siRNA conjugate4.);

FIG. 9 shows an electrophoresis photograph expressing the degrees ofsiRNA degradation according to the time in the presence of serumprotein, in order to evaluate the stability in the blood of a nakedsiRNA in which none of polymer compounds are conjugated, andsiRNA-polymer compound conjugates in which a hydrophilic polymercompound, PEG is conjugated;

FIG. 10 is a schematic diagram of a nanoparticle formed by asiRNA-polymer compound conjugate;

FIG. 11 shows particle size results of nanoparticles consisting of nakedsiRNAs in which none polymer compounds are conjugated, measured by thezeta-potential measuring instrument;

FIG. 12 shows size results of nanoparticles each consisting ofsiRNA-polymer compound conjugates 9, measured by the zeta-potentialmeasuring instrument;

FIG. 13 shows size results of nanoparticles each consisting ofsiRNA-polymer compound conjugates 10, measured by the zeta-potentialmeasuring instrument;

FIG. 14 shows size results of nanoparticles each consisting ofsiRNA-polymer compound conjugates 11, measured by the zeta-potentialmeasuring instrument;

FIG. 15 shows size results of nanoparticles each consisting ofsiRNA-polymer compound conjugates 12, measured by the zeta-potentialmeasuring instrument;

FIG. 16 shows size results of nanoparticles each consisting ofsiRNA-polymer compound conjugates 13, measured by the zeta-potentialmeasuring instrument;

FIG. 17 is a graph comparing mRNA expression degrees of survivin geneafter transfection together with a transfection reagent, in order toanalyze RNAi effects of a naked siRNA and respective siRNA-polymercompound conjugates in which a hydrophilic polymer compound, PEG, isconjugated;

FIG. 18 is a graph comparing mRNA expression degrees of survivin geneafter transfection together with a transfection reagent, in order toanalyze RNAi effects of a naked siRNA and respective long-sequencesiRNAs transformed in the siRNA-polymer compound conjugate 4; and

FIG. 19 is a graph comparing mRNA expression degrees of survivin geneafter transfection in absence of a transfection reagent, in order toanalyze RNAi effects of a naked siRNA and siRNA-polymer compoundconjugates 1 to 5 and 9 to 14.

BEST MODE

Hereinafter, the exemplary embodiments of the present invention will bedescribed in detail. However, the following exemplary embodimentsdescribe the present invention by way of example only but are notlimited thereto.

Example 1 Preparation of Solid Support for Preparing 3′-PEGOligonucleotide Example 1-1 Preparation of 3′-PEG Reagents (Compounds A,B, and C) for Binding with LCAA-CPG

In the subsequent example, 3′-PEG-CPG was prepared as shown in thefollowing reaction formula.

Example 1-1-1 Preparation of 2-[bis-(4-dimethoxytrityl)-poly(ethyleneglycol)]

30 g (15 mmol) of polyethylene glycol 2000 (Alfa Aesar GmbH & Co. KG,Germany), as a starting material, was dissolved in 270 ml of pyridine(Sigma Aldrich, USA), followed by addition of 3.55 ml (25.5 mmol) oftriethylamine (Sigma Aldrich, USA) and 7.12 g (21 mmol) of4,4′-dimethoxy trityl chloride (GL biochem, China), and then theresultant substance was reacted at room temperature for 20 hours. Thereactant mixture after completion of reaction was concentrated, andextracted with 450 ml of ethyl acetate and 450 ml of water, followed byvacuum evaporation and then vacuum drying, to obtain2-[bis-(4-dimethoxytrityl)-poly(ethylene glycol) 23 g (66%).

¹H NMR data of the compound are shown in FIG. 2.

¹H NMR (CDCl₃); δ 1.93 (br, 1, OH), 3.20-3.80 (m, 186, PEG, DMT-OCH₃),6.80-6.83 (m, 4, DMT), 7.19-7.47 (m, 9, DMT)

Example 1-1-2 Preparation of succinic acid2-[bis-(4-dimethoxytrityl)-poly(ethyleneglycol)] [Compound A]

3.9 g (1.672 mmol) of 2-[bis-(4-dimethoxytrityl)-poly(ethyleneglycol)]obtained in the example 1-1-1 was dissolved in 20 ml of pyridine, andthen cooled to 0° C. 351 mg (3.512 mmol) of succinic acid anhydride(Acros Organics, USA) and 42.5 mg (0.334 mmol) of DMAP(4-dimethylaminopyridine, Sigma Aldrich, USA) were added to the reactantsubstance, and stirred at 50° C. for 3 days, and then the reaction wasfinished. The reactant mixture after completion of reaction wasvacuum-evaporated to obtain succinic acid2-[bis-(4-dimethoxytrityl)-poly(ethylene glycol)] [Compound A] 3.65 g(90%, white solid).

¹H NMR data of the compound are shown in FIG. 3.

¹H NMR (CDCl₃); δ 2.65 (m, 2, CH₂CO), 3.20-3.88 (m, 186, PEG, DMT-OCH₃),4.25 (m, 2, CH₂CO), 6.80-6.82 (m, 4, DMT), 7.19-7.47 (m, 9, DMT).

Example 1-1-3 Preparation of para-nitrophenyl succinic acid2-[bis-(dimethoxytrityl)-poly(ethylene glycol))] [Compound B]

1 g (0.411 mmol) of the compound A obtained in the example 1-1-2 wasdissolved in 20 ml of methylene chloride (DaeYeon Chemicals, Co. Ltd.,Korea), and cooled to 0° C. 143 μl (1.03 mmol) of triethylamine was putinto the reactant substance, and 149 mg (0.740 mmol) of 4-nitro phenylchloroformate was added thereto. Then, the temperature was raised toroom temperature and the resultant substance was stirred for 4 hours,and then the reaction was finished. The reactant mixture aftercompletion of reaction was once washed with 20 ml of aqueous saturatedNaHCO₃ and 20 ml of 1M citric acid (Sigma Aldrich, USA) which was cooledto 0° C.˜4° C., and then dried with Na₂SO₄ (Samchum Chemical Co.,Korea). The resultant substance was filtered by using a filtering flask,a Buchner funnel, or an aspirator, followed by vacuum evaporation, toobtain para-nitrophenylsuccinic acid2-[bis-(4-dimethoxytrityl)-poly(ethylene glycol)[Compound B] 1.0 g (94%,creamy solid).

¹H NMR data of the compound are shown in FIG. 4.

¹H NMR (CDCl₃); δ 2.80-2.90 (m, 2, CH₂CO), 3.20-3.87 (m, 186, PEG,DMT-OCH₃), 4.25 (m, 2, CH₂CO), 6.80-6.82 (m, 4, DMT), 7.19-7.47 (m, 9,DMT)

Example 1-1-4 Preparation of 2,5-dioxo-pyrolidine-1-ylester succinicacid 2-[bis-(4-dimethoxytrityl)-poly(ethylene glycol)] [Compound C]

500 mg (0.206 mmol) of the compound A obtained in the example 1-1-2 wasdissolved in 10 ml of methylene chloride, and then 83.14 μl (1.03 mmol)of pyridine was put thereinto. 165 mg (0.781 mmol) of N-succinimidyltrifluoro acetic acid (Sigma Aldrich, USA) was added thereto, andstirred at room temperature for 7 hours, and then the reaction wasfinished. The reactant mixture after completion of reaction was vacuumevaporated, to obtain 2,5-dioxo-pyrrolidin-1-yl ester succinic acid2-[bis-(4-dimethoxytrityl)-poly(ethyleneglycol)] [Compound C] 490 mg(94%, white solid).

¹H NMR data of the compound are shown in FIG. 5.

¹H NMR (CDCl₃); δ 2.72-2.97 (m, 6, CH₂CO, CH₂CH₂), 3.20-3.87 (m, 186,PEG, DMT-OCH₃), 4.27-4.28 (m, 2, CH₂CO), 6.80-6.83 (m, 4, DMT),7.20-7.47 (m, 9, DMT)

Example 1-2 Binding of LCAA-CPG and 3′-PEG Reagent (Compound A)

In the subsequent example, CPG and 3′-PEG reagent were bound as shown inthe following reaction formula:

Example 1-2-1 Preparation of LCAA-CPG (2000 Å)

10 g of CPG (Silicycle Inc., Canada) having a diameter of 40˜75 μm and ananopore of 2000 Å was equally mixed and wet with 100 μm of toluene, andthen 2 ml of 3-aminopropyltriethoxysilane (TCI Org. Chem, Japan) was putthereinto. Then, the resultant substance was mixed and then reacted atroom temperature for 8 hours. The mixture after completion of reactionwas filtered, and washed with methanol, water, and methylene chloride inthat order, followed by vacuum drying, to obtain 10 g of LCAA-CPG (2000Å).

Example 1-2-2 Preparation of 3′-PEG-CPG (2000 Å) using succinic acid2-[bis-(4-dimethoxytrityl)-poly(ethylene glycol)] [Compound A]

2 g of LCAA-CPG (2000 Å) obtained in the example 1-2-1 was wet in 20 mlof methylene chloride. In addition, the LCAA-CPG (2000 Å) solution wasequally mixed with a solution in which 80 mg of the compound A, 14 μl ofTEA (triethylamine, Sigma Aldrich, USA). 15 mg of BOP(benzortiazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate,TCI Org. Chem, Japan), and 5 mg of HOBT (1-Hydroxybenzotriazoleanhydrous, TCI Org. Chem, Japan) were dissolved in 2 ml of methylenechloride. The resultant substance was reacted at reflux for 8 hours, andthen the mixture after completion of reaction was filtered and washedwith methanol, water, and methylene glycol in that order, followed byvacuum drying.

1 g of the resultant substance was wet in 10 ml of pyridine, and then 1ml of 1-methylimidazole (Sigma Aldrich, USA) and 1.6 ml of aceticanhydride (Sigma Aldrich, USA) were put thereinto. The resultantsubstance was equally mixed, and reacted at room temperature for 8hours. The capping-completed CPG obtained after completion of reactionwas washed with methanol, water, methanol, and methylene chloride inthat order, followed by vacuum drying, to obtain 1 g of 3′-PEG-CPG.

Example 1-3 Binding of LCAA-CPG (2000 Å) and 3′-PEG Reagent (Compound B)

Preparation of 3′-PEG-CPG (2000 Å) was performed by using the compoundB.

Specifically, 1 g of LCAA-CPG (2000 Å) obtained in the example 1-2-1 wassufficiently wet in 8 ml of pyridine. In addition, a solution in which205 mg (2eq) of the compound B and 55 μl of triethylamine were dissolvedin 2 ml of pyridine was equally mixed with the LCAA-CPG solution. Theresultant substance was reacted at 50˜60° C. for 8 hours, and then themixture after completion of reaction was filtered. The filteredcoupling-CPG was washed with methanol, water, and methylene chloride inthat order, followed by vacuum drying. 1 g of the coupling-CPG aftercompletion of drying was wet in 10 ml of pyridine, and then 500 μl of1-methyl imidazole and 800 μl of acetic anhydride were added thereto.The resultant substance was equally mixed, and then reacted at roomtemperature for 8 hours. The mixture after completion of reaction wasfiltered, and then the coupling-CPG was washed with methanol, water, andmethylene chloride in that order, followed by vacuum drying, to obtain3′-PEG-CPG 1 g.

FIG. 6 shows Maldi-Tof molecular weight determination results of siRNAsprepared by using 3′-PEG-CPG as a starting material, as shown in theexample 2 to be described later.

3′-PEG-CPG preparation sequence;

sense

(Sequence ID No. 1) 5′-AAGGAGAUCAACAUUUUCA(dTdT)-PEG(6664.96Da + 2000Da)antisense (Sequence ID No. 5)5′-UGAAAAUGUUGAUCUCCUU(dTdT)-PEG(6592.84Da + 2000Da)

It could be found that Maldi-Tof molecular weight has increased by themolecular weight (2000 Da) of PEG.

Example 1-4 Binding of LCAA-CPG (2000 Å) and 3′-PEG Reagent (Compound C)

Preparation of 3′-PEG-CPG (2000 Å) was performed by using the compoundC.

Specifically, 1 g of LCAA-CPG (2000 Å) obtained in the example 1-2-1 wassufficiently wet in 8 ml of pyridine. In addition, a solution in which200 mg of the compound C and 55 μl of triethylamine were dissolved in 2ml of pyridine was equally mixed with the LCAA-CPG solution. Theresultant substance was reacted at 50˜60° C. for 8 hours, and then themixture after completion of reaction was filtered. The filteredcoupling-CPG was washed with methanol, water, and methylene chloride inthat order, followed by vacuum drying. 1 g of the coupling-CPG aftercompletion of drying was wet in 10 ml of pyridine, and then 500 μl of1-methyl imidazole and 800 μl of acetic anhydride were added thereto.The resultant substance was equally mixed, and then reacted at roomtemperature for 8 hours. The capping-completed CPG after completion ofreaction was washed with methanol, water, and methylene chloride in thatorder, followed by vacuum drying, to obtain 3′-PEG-CPG 1 g.

FIG. 7 shows results of siRNAs prepared by using 3′-PEG-CPG as astarting material, as shown in the example 2 to be described later.

3′-PEG-CPG preparation sequence;

sense

(Sequence ID No. 1) 5′-AAGGAGAUCAACAUUUUCA(dTdT)-PEG(6664.96Da + 2000Da)antisense (Sequence ID No. 5)5′-UGAAAAUGUUGAUCUCCUU(dTdT)-PEG(6592.84Da + 2000Da)

It could be found that Maldi-Tof molecular weight has increased by themolecular weight (2000 Da) of PEG.

Example 2 Preparation of siRNA-Polymer Compound Conjugates

In the following examples, survivin siRNA was used in order to suppresssurvivin. The survivin is a protein expressed commonly in mostneoplastic tumors or transformed cell lines, tested until now, and thusit is expected to become an important target in anticancer treatment(Abbrosini G. et al. Nat. Med. 3(8): 917-921, 1997). A survivin siRNAsequence of the present invention, when composed of 19 nucleotides,consists of a sense strand of the Sequence ID No. 1 and an antisensestrand having a sequence complementary to the sense strand, and besidethis, when composed of 23, 27, or 31 nucleotides, has a base sequence ofthe Sequence ID No. 2, 3, or 4.

(Ssequence ID No. 1.) 5′-AAGGAGAUCAACAUUUUCA-3′ (Ssequence ID No. 2)5′-AGGAAAGGAGAUCAACAUUUUCA-3′ (Sequence ID No. 3)5′-AGGAAAGGAGAUCAACAUUUUCAAAUU-3′ (Sequence ID No. 4)5′-AAAGGAGAUCAACAUUUUCAAAUUAGAUGUU-3′

The siRNA was prepared by linking phosphordiester bonds building an RNAbackbone structure, using β-cyanoethyl phosphoramidite (Shina et al.Nucleic Acids Research, 12:4539-4557, 1984). Specifically, a series ofprocedures consisting of deblocking, coupling, oxidation and cappingwere repeatedly performed on a sold support on which nucleotide wasattached, by using an RNA synthesizer (384 Synthesizer, BIONEER, Korea),to obtain the reactant containing a desired length of RNA.

Additively, the siRNA-polymer compound conjugate was prepared by linkingPEG to a 5′-end region, or hexadecane (C16) or octadecane (C18)saturated hydrocarbon, to the 5′-end region by using a dodecane linker,which is a hydrophobic polymer compound. In addition, theabove-mentioned reaction was performed by using 3′PEG-CPG prepared inthe example 1 as a support, to obtain the siRNA-polymer compoundconjugate in which PEG was provided to 3′-end region.

It was identified whether the reactant substances were consistent with anucleotide sequence which is to be prepared, by separating RNA from thereactant substances using an HPLC (LC-20A Prominence, SHIMADZU, Japan)and measuring the molecular weight thereof using an MALDI-TOF massspectrometer (MALDI TOF-MS, SHIMADZU, Japan). After that, a sense RNAstrand and an antisense RNA strand were mixed in the same amount, andput into a 1× annealing buffer (30 mM HEPES, 100 Mm potassium acetate, 2mM magnesium acetate, pH 7.0˜7.5). The resultant substance was reactedin a constant temperature bath of 90° C. for 3 minutes, and then againreacted at 37° C., to prepare a double-stranded siRNA-polymer compoundconjugate. The prepared siRNA-polymer compound conjugates havestructures shown in Table 1. Annealing of the prepared siRNA-polymercompound conjugates was confirmed through electrophoresis photographs(FIG. 8).

TABLE 1 Structures and end modification types of siRNA-polymer compoundconjugates Structure names End modification Conjugate names ofconjugates types siRNA naked siRNA Sense: none Antisense: nonesiRNA-polymer 5′PEG-sense siRNA Sense: 5′PEG compound conjugate 1Antisense: none siRNA-polymer 5′PEG-antisense Sense: none compoundconjugate 2 siRNA Antisense: 5′PEG siRNA-polymer 5′ssPEG-antisenseSense: none compound conjugate 3 siRNA Antisense: 5′ssPEG siRNA-polymer5′PEG + PEG siRNA Sense: 5′PEG compound conjugate 4 Antisense: 5′PEGsiRNA-polymer 5′PEG + ssPEG siRNA Sense: 5′PEG compound conjugate 5Antisense: 5′ssPEG siRNA-polymer 3′PEG-sense siRNA Sense: 3′PEG compoundconjugate 6 Antisense: none siRNA-polymer 3′PEG-antisense Sense: nonecompound conjugate 7 siRNA Antisense: 3′PEG siRNA-polymer 3′PEG + PEGsiRNA Sense: 3′PEG compound conjugate 8 Antisense: 3′PEG siRNA-polymer5′C18-sense siRNA Sense: 5′C18-C6- compound conjugate 9 ss-C6 Antisense:none siRNA-polymer 5′C18 + PEG siRNA Sense: 5′C18-C6- compound conjugatess-C6 10 Antisense: 5′PEG siRNA-polymer 5′C16 + PEG siRNA Sense:5′C16-C6- compound conjugate ss-C6 11 Antisense: 5′PEG siRNA-polymer5′C18-antisense Sense: none compound conjugate siRNA Antisense: 5′C18-12 C6-ss-C6 siRNA-polymer 5′PEG + C18 siRNA Sense: 5′PEG compoundconjugate Antisense: 5′C18- 13 C6-ss-C6 siRNA-polymer 5′PEG + C16 siRNASense: 5′PEG compound conjugate Antisense: 5′C16- 14 C6-ss-C6 * In thestructures of conjugates, “ss” means a disulfide bond, and “C16” or“C18” represents C16 or C18 hydrocarbon. Therefore, “C18-C6-ss-C6” and“C16-C6-ss-C6” mean hydrophobic polymer compounds.

Example 3 Evaluation on Stability of siRNA-Polymer Compound ConjugatesIn Vivo Conditions

It was identified whether or not the siRNA-polymer compound conjugatesprepared and separated in the Example 2 have improved stability comparedwith a naked siRNA in which none of polymer compound is bound. The nakedsiRNA without modification and the siRNA-polymer compound conjugates 1to 5 prepared in the Example 2 were incubated for 0, 1, 3, 6, 9, 12, 24,36, or 48 hours, in a culture medium containing 10% fetal bovine serum(FBS), which imitates in vivo conditions, and then the degrees to whichthe siRNA was degraded were evaluated by using electrophoresis.

The results showed that siRNA-polymer compound conjugates having PEGintroduced therein exhibited siRNA stability for up to 48 hours (FIG.9). The siRNA stability was exhibited for 12 hours to 24 hours evenunder the condition of 100% serum.

Example 4 Measurement on Sizes of Nanoparticles of siRNA-HydrophobicPolymer Compound Conjugates

In each case of siRNA-polymer compound conjugates 9 to 14, ananoparticle consisting of siRNA-polymer compound conjugates, that is tosay, a micelle is formed by hydrophobic interaction between hydrophobicpolymer compounds provided at ends of the siRNAs (FIG. 10). The sizes ofthe nanoparticles were measured using a zeta-potential measuringinstrument. The sizes of nanoparticles consisting of the respectivesiRNA-polymer compound conjugates 9 to 13 prepared in the Example 2, andsiRNAs were measured.

Specifically, 2 nmol of siRNA and the siRNA-polymer compound conjugateswere dissolved in 1 ml of distilled water, and then the nanoparticlesthereof was homogenized (200 W, 40 kHz, 5 sec) by using an ultrasonichomogenizer (Wiseclean, DAIHAN, Korean). The sizes of the homogenizednanoparticles were measured by using the zeta-potential measuringinstrument (Nano-ZS, MALVERN, UK). Here, the refractive index andabsorption index for materials were set to 1.454 and 0.001,respectively, and the temperature of water as a solvent, 25° C., wasinput, and the viscosity and refractive index thereof were input. Aone-time measurement consists of 20 repetitive size measurements, andthis measurement was performed three times.

FIG. 11 shows size results of naked siRNA nanoparticles, measured by thezeta-potential measuring instrument. It showed that sizes of 142˜295 nm(maximum point: 164 nm) account for 73.5% of total nanoparticles eachconsisting of siRNAs.

FIG. 12 shows size results of nanoparticles each consisting ofsiRNA-polymer compound conjugate 9, measured by the zeta-potentialmeasuring instrument. It showed that sizes of 4.19˜7.53 nm (maximumpoint: 6.50 nm) account for 59.1% of total nanoparticles each consistingof siRNA-polymer compound conjugate 9.

FIG. 13 shows size results of nanoparticles each consisting ofsiRNA-polymer compound conjugate 10, measured by the zeta-potentialmeasuring instrument. It showed that sizes of 5.61˜10.1 nm (maximumpoint: 8.72 nm) account for 58.9% of total nanoparticles each consistingof siRNA-polymer compound conjugate 10.

FIG. 14 shows size results of nanoparticles each consisting ofsiRNA-polymer compound conjugate 11, measured by the zeta-potentialmeasuring instrument. It showed that sizes of 5.61˜10.1 nm (maximumpoint: 8.72 nm) account for 45.6% of total nanoparticles each consistingof siRNA-polymer compound conjugate 11.

FIG. 15 shows size results of nanoparticles each consisting ofsiRNA-polymer compound conjugate 12, measured by the zeta-potentialmeasuring instrument. It showed that sizes of 4.85˜5.61 nm account for23.6%, sizes of 21.0˜32.7 nm accounts for 23.5%, and sizes of 68.1˜78.8nm accounts for 23.1% of total nanoparticles each consisting ofsiRNA-polymer compound conjugate 12.

FIG. 16 shows size results of nanoparticles each consisting ofsiRNA-polymer compound conjugate 13, measured by the zeta-potentialmeasuring instrument. It showed that sizes of 4.85˜8.72 nm (the maximumpoint: 5.61 nm) account for 84.6% of total nanoparticles each consistingof siRNA-polymer compound conjugate 13.

In cases of siRNA-polymer compound conjugates 9 to 13 except for thesiRNA-polymer compound conjugate 12, the sizes of the nanoparticles weremostly 4˜8 nm. In the case of the siRNA-polymer compound conjugate 12,the sizes of nanoparticles were variously measured, the reason of whichis considered that respective nanoparticles aggregated as time passesduring the measuring process, even though homogenization is performedusing the ultrasonic homogenizer. As shown in FIGS. 12 to 16, themeasured sizes of nanoparticles each consisting of siRNA conjugatesexhibit 100 nm or less, which are sufficient sizes to be endocytosedinto cells through pinocytosis (Kenneth A. Dawson et al. naturenanotechnology 4:84-85, 2009).

Example 5 Inhibition of Expression of Target Genes in Tumor Cell Linesby Using siRNA-Polymer Compound Conjugates with Transfection Reagents

Human cervical cancer cell lines, which are tumor cell lines, wererespectively transfected with siRNA-polymer compound conjugates 1 to 8prepared in the Example 2, and expression levels of survivin gene in thetransfected tumor cell lines were analyzed.

Example 5-1 Culture of Tumor Cell Lines

Human cervical cancer cells (HeLa), obtained from American Type CultureCollection (ATCC), were cultured in an RPMI 1640 culture medium (GIBCO,Invitrogen, USA), in which 10%(v/v) fetal bovine serum, penicillin 100units/ml, and streptomycin 100 μg/ml were added at 37° C. under thecondition of 5%(v/v) CO₂.

Example 5-2 Inhibition of Expression of Target Gene by UsingsiRNA-Polymer Compound Conjugates

HeLa tumor cell lines were transfected with siRNA-polymer compoundconjugates 1 to 8 of Sequence ID No. 1, prepared in the Example 2, andexpression of survivin genes in the transfected tumor cell lines wereanalyzed.

Example 5-2-1 Transfection of Tumor Cell Lines by Using siRNA-PolymerCompound Conjugates

1.3×10⁵ tumor cell lines cultured in the Example 5-1 were cultured inthe RPMI 1640 medium within a 6-well plate at 37° C. for 18 hours underthe condition of 5%(v/v) CO₂, followed by removal of the medium, andthen 800 μl of the Opti-MEM medium (GIBCO, USA) was dispensed for eachwell.

Meanwhile, 2 μl of Lipofectamine™ 2000 (Invitrogen, USA) and 198 μl ofOpti-MEM medium were mixed, followed by reaction therebetween at roomtemperature for 5 minutes, and then 0.8 or 4 μl of the respectivesiRNA-polymer compound conjugates (25 pmole/μl) prepared in the Examples2 were added thereto (finally treated at 20 or 100 nM). Then, thisresultant substance was again reacted at room temperature for 20minutes, to prepare a solution.

After that, 200 μl of the transfection solution was dispensed to each ofthe wells in which the Opti-MEM medium had been dispersed, and the tumorcells were cultured for 6 hours, followed by removal of the Opti-MEMmedium. 2.5 ml of the RPMI 1640 culture medium is dispensed thereto, andthen the tumor cells were cultured at 37° C. under the condition of5%(v/v) CO₂ for 24 hours.

Example 5-2-2 Relative Quantitative Analysis of Survivin Gene mRNA

Total RNA was extracted from the cell line transfected in the example5-2-1 to prepare cDNA, and then the quantity of the survivin gene mRNAwas relatively quantitated through the realtime PCR.

Example 5-2-2-1 Separation of RNA and Preparation of cDNA from theTransfected Cells

Total RNA was extracted from the cell line transfected in the example5-2-1 by using an RNA extraction kit (AccuPrep Cell total RNA extractionkit, BIONEER, Korea), and cDNA was prepared from the extracted RNA byusing an RNA reverse transcriptase (AccuPower CycleScript RTPremix/dT₂₀, BIONEER, Korea), as follows.

Specifically, 1 μg of the extracted RNA was put in each of 0.25 mlEppendorf tubes containing AccuPower CycleScript RT Premix/dT20(BIONEER, Korea), and the distilled water treated with diethylpyrocarbonate (DEPC) was added thereto to have a total volume of 20 μl.By using a PCR machine (MyGenie™ 96 Gradient Thermal Block, BIONEER,Korea), two steps of RNA-primer hybridization at 30° C. for 1 minute andsynthesis of cDNA at 52° C. for 4 minutes were repeated six times. Theninactivation of enzyme was performed at 95° C. for 5 minutes to finishthe amplification reaction.

Example 5-2-2-2 Relative Quantitative Analysis of Survivin Gene mRNA

The relative quantity of the survivin mRNA was quantitated through therealtime PCR by using the cDNA prepared in the example 5-2-2-1 as atemplate as follows.

That is to say, the cDNA prepared in the example 5-2-2-1 was ⅕-dilutedwith the distilled water in each well of a 96-well plate, and then 3 μlof the diluted cDNA, 10 μl of 2× GreenStar™ PCR master mix (BIONEER,Korea), 6 μl of distilled water, and 1 μl of survivin qPCR primer (10pmole/μl each, BIONEER, Korea) were input to prepare a mixture liquid inorder to analyze the survivin expression level. On the other hand, byusing HMBS (Hydroxymethylbilane synthase), HPRT1 (Hypoxanthinephosphoribosyl-transferase1), UBC (Ubiquitin C), and YWHAZ (Tyrosine3-monooxygenase/tryptophan 5-monooxygenase activation protein, zetapolypeptide), which are housekeeping genes (hereafter, referred to as“HK genes”), as the reference gene, in order to normalize the mRNAexpression level, the cDNA prepared in the example 5-2-2-1 was⅕-diluted, and then 3 μl of the diluted cDNA, 10 μl of 2× GreenStar™ PCRmaster mix (BIONEER, Korea), 6 μl of distilled water, and 1 μl of qPCRprimer of each HK gene (10 pmole/μl each, BIONEER, Korea) were input toprepare a HK gene realtime PCR mixture liquid in each well of the96-well plate. The following reaction was performed on the 96-well platecontaining the mixture liquid by using an Exicycler™ 96 Real-TimeQuantitative Thermal Block (BIONEER, Korea).

Enzyme activation and secondary structure of cDNA were removed by thereaction at 95° C. for 15 minutes. Then, four steps of denaturing at 94°C. for 30 seconds, annealing at 58° C. for 30 seconds, extension at 72°C. for 30 seconds, and SYBR green scan, were repetitively performed 42times, and then the final extension at 72° C. for 3 minutes wasperformed. Then, the temperature was kept at 55° C. for 1 minute, and amelting curve of 55° C.˜95° C. was analyzed.

After finishing the PCR, the survivin Ct (threshold cycle) valuesobtained respectively were corrected by using the mRNA values(normalization factor, NF) normalized through the HK genes, and then ΔCtvalues were obtained between the Ct value of a control group treatedwith only a transfection reagent and the corrected Ct values. Theexpression rates of survivin mRNA were compared with one another byusing the ΔCt values and the calculation equation of 2^((−ΔCt))×100(FIG. 17). In FIG. 17, mock means the control group treated with onlythe transfection reagent.

As a result, as shown in FIG. 17, it showed that RNAi effect of thesiRNA was varied depending on end modification types of thesiRNA-polymer compound conjugates in which PEG, the hydrophilic polymercompound was conjugated. Particularly, the conjugates 6 to 8 each havingthe end modification type in which PEG was conjugated to the 3′-endregion, exhibited expression-inhibiting degrees similar to that of thenaked siRNA. Therefore, the conjugates 6 to 8 are expected to have alittle steric hindrance in forming a complex with an RNA-inducedsilencing complex (RISC) on the RNAi mechanism of the siRNA. Inaddition, most siRNA-PEG conjugates exhibited higher inhibition oftarget gene mRNA expression in a low concentration (20 nM) treatmentcondition than in a high concentration (100 nM) treatment condition, andthus it is expected that siRNA is prevented from being bound with theRISC due to the PEG as the concentration condition of the siRNA-PEGconjugate is higher.

Example 5-3 Inhibition of Expression of Target Gene by UsingLong-Sequence siRNA-Polymer Compound Conjugates

When the cells were transfected with siRNA-hydrophilic polymer compoundconjugates together with a transfection reagent, inhibition of targetgene mRNA expression was analyzed. Here, the siRNAs, in which endmodification into the structure of siRNA-polymer compound conjugate 4was induced for each base sequence of siRNA Sequence ID No. 1 to 4, wereused.

Example 5-3-1 Transfection of Tumor Cell Lines by Using siRNA-PolymerCompound Conjugates

1.3×10⁵ tumor cell lines cultured in the Example 5-1 were cultured inthe RPMI 1640 medium within a 6-well plate at 37° C. for 24 hours underthe condition of 5%(v/v) CO₂, followed by removal of the medium, andthen 800 μl of the Opti-MEM medium was dispensed for each well.

Meanwhile, 2 μl of Lipofectamine™ 2000 and 198 μl of the Opti-MEM mediumwere mixed, followed by reaction therebetween at room temperature for 5minutes, and then 0.8 or 4 μl of the respective siRNA-polymer compoundconjugates (25 pmole/μl) prepared in the Examples 2 were added thereto(finally treated at 20 or 100 nM). Then, this resultant substance wasagain reacted at room temperature for 20 minutes, to prepare a solution.

After that, 200 μl of a transfection solution was dispensed to each ofthe wells in which the Opti-MEM medium had been dispersed, and the tumorcells were cultured for 6 hours, followed by removal of the Opti-MEMmedium. 2.5 ml of the RPMI 1640 culture medium is dispensed thereto, andthen the tumor cells were cultured at 37° C. under the condition of5%(v/v) CO₂ for 24 hours.

Example 5-3-2 Relative Quantitative Analysis of Survivin Gene mRNA

Total RNA was extracted from the cell line transfected in the example5-3-1 to prepare cDNA, and then the quantity of survivin gene mRNA wasrelatively quantitated through the real-time PCR.

Example 5-3-2-1 Separation of RNA and Preparation of cDNA from theTransfected Cells

Total RNA was extracted from the cell line transfected in the example5-3-1 by using an RNA extraction kit (AccuPrep Cell total RNA extractionkit, BIONEER, Korea), and cDNA was prepared from the extracted RNA byusing an RNA reverse transcriptase (AccuPower CycleScript RTPremix/dT₂₀, BIONEER, Korea), as follows.

Specifically, 1 μg of the extracted RNA was put into each of 0.25 mlEppendorf tubes containing AccuPower CycleScript RT Premix/dT20(BIONEER, Korea), and the distilled water treated with diethylpyrocarbonate (DEPC) was added thereto to have a total volume of 20 μl.By using a PCR machine (MyGenie™ 96 Gradient Thermal Block, BIONEER,Korea), two steps of RNA-primer hybridization at 30° C. for 1 minute andpreparation of cDNA at 52° C. for 4 minutes were repeated six times.Then inactivation of enzyme was performed at 95° C. for 5 minutes tofinish the amplification reaction.

Example 5-3-2-2 Relative Quantitative Analysis of Survivin Gene mRNA

The relative quantity of the survivin gene mRNA was quantitated throughthe realtime PCR by using the cDNA prepared in the example 5-3-2-1 as atemplate as follows.

That is to say, the cDNA prepared in the example 5-3-2-1 was ⅕-dilutedin each well of a 96-well plate, and then 3 μl of the diluted cDNA, 10μl of 2× GreenStar™ PCR master mix (BIONEER, Korea), 6 μl of distilledwater, and 1 μl of survivin qPCR primer (10 pmole/μl each, BIONEER,Korea) were input to prepare a mixture liquid in order to analyze thesurvivin expression level. On the other hand, by using HMBS, HPRT1, UBC,and YWHAZ, which are HK gene, as the reference gene, in order tonormalize the mRNA expression level, the cDNA prepared in the example5-3-2-1 was ⅕-diluted, and then 3 μl of the diluted cDNA, 10 μl of 2×GreenStar™ PCR master mix (BIONEER, Korea), 6 μl of distilled water, and1 μl of qPCR primer of each HK gene (10 pmole/μl each, BIONEER, Korea)were input to prepare a HK gene realtime PCR mixture liquid in each wellof the 96-well plate. The following reaction was performed on the96-well plate containing the mixture liquid by using an Exicycler™ 96Real-Time Quantitative Thermal Block (BIONEER, Korea).

Enzyme activation and secondary structure of cDNA were removed by thereaction at 95° C. for 15 minutes. Then, four steps of denaturing at 94°C. for 30 seconds, annealing at 58° C. for 30 seconds, extension at 72°C. for 30 seconds, and SYBR green scan were repetitively performed 42times, and then the final extension at 72° C. for 3 minutes wasperformed. Then the temperature was kept at 55° C. for 1 minute, and amelting curve of 55° C.˜95° C. was analyzed.

After finishing the PCR, the survivin Ct (threshold cycle) valuesobtained respectively were corrected by using the mRNA values(normalization factor, NF) normalized through the HK genes, and then ΔCtvalues were obtained between the Ct value of a control group treatedwith only the transfection reagent and the corrected Ct values. Theexpression rates of survivin mRNA were compared with one another byusing the ΔCt values and the calculation equation of 2^((−ΔCt))×100(FIG. 18). In FIG. 18, mock means the control group treated with onlythe transfection reagent, and 19mer, 23mer, 27mer, and 31mer representSequence ID No. 1 to 4, respectively. 5′P+P represents a structure ofthe siRNA-polymer compound conjugate 4. The cells were treated with 20nM and 100 nM respectively, and the inhibition degrees of the targetgene expression were compared with one another.

As a result, as shown in FIG. 18, the long-chain naked siRNAstransformed in the form of siRNA-polymer compound conjugate 4, exhibitedless difference in inhibition of the target gene mRNA expression,compared with the naked siRNA. Therefore, it could be found that thetransformed long-chain siRNA decreases the steric hindrance phenomenondue to PEG compared with a shot chain.

That is to say, in a case of the long-chain siRNA, the siRNA is cleavedin a structure of 19+2 by a dicer in an operation mechanism of RNAi, andthe cleaved siRNA is bound to an RISC complex to cause the operationmechanism of RNAi. For this reason, the long-chain siRNA, in which PEGis provided at both end regions, causes existence of a large quantity ofsiRNAs without PEG attachment, and thus has a relatively highinteraction with the RISC complex, compared with the Sequence ID No. 1,which is believed to maintain the RNAi induction effect.

Example 6 Inhibition of Expression of Target Gene in Tumor Cell Lines byUsing Only siRNA-Polymer Compound Conjugates without TransfectionReagents

HeLa tumor cell lines were transfected with siRNA-polymer compoundconjugates 1 to 14 prepared in the Example 2, and expression of survivingenes of the transfected tumor cell lines was analyzed.

Example 6-1 Culture of Tumor Cell Lines

Human uterine cancer cells (HeLa), obtained from American Type CultureCollection (ATCC), were cultured in an RPMI 1640 culture medium(GIBCO/Invitrogen, USA), in which 10%(v/v) fetal bovine serum,penicillin 100 units/ml, and streptomycin 100 μg/ml were added, at 37°C. under the conditions of 5%(v/v) CO₂.

Example 6-2 Transfection of Tumor Cell Lines by Using siRNA-PolymerCompound Conjugates

1.3×10⁵ tumor cell lines cultured in the Example 6-1 were cultured inthe RPMI 1640 medium in a 6-well plate at 37° C. for 24 hours under thecondition of 5%(v/v) CO₂, followed by removal of the medium, and then900 μl of the Opti-MEM medium was dispensed for each well.

Meanwhile, 100 μl of the Opti-MEM medium, 5 or 10 μl of the respectivesiRNA-polymer compound conjugates 1 to 5 (1 nmole/μl) prepared in theexample 2 were added thereto (finally treated at 500 nM or 1 μM), andthe resultant substance was again reacted at room temperature for 20minutes, to prepare the solution.

Meanwhile, 100 μl of the Opti-MEM medium, 5 or 10 μl of the respectivesiRNA-polymer compound conjugates 9 to 14 (1 nmole/μl) prepared in theexample 2 were added thereto (finally treated 500 nM or 1 μM), andmicelles consisting of siRNA-hydrophobic polymer compound conjugateswere homogenized through sonication by high frequency sounds, to preparethe solution.

After that, 100 μl of the transfection solution was dispensed to each ofthe wells in which the Opti-MEM medium had been dispersed, and the tumorcells were cultured for 24 hours, followed by addition of 1 ml of RPMI1640 medium containing 20% FBS. The cells were further cultured at 37°C. for 24 hours under the condition of 5%(v/v) CO₂, treated withsiRNA-polymer compound conjugates, and then cultured for total 48 hours.

Example 6-3 Relative Quantitative Analysis of Survivin Gene mRNA

Total RNA was extracted from the cell line transfected in the example6-2 to prepare cDNA, and then the quantity of survivin gene mRNA wasrelatively quantitated through the real-time PCR.

Example 6-3-1 Separation of RNA and Preparation of cDNA from theTransfected Cells

Total RNA was extracted from the cell line transfected in the example6-2 by using an RNA extraction kit (AccuPrep Cell total RNA extractionkit, BIONEER, Korea), and cDNA was prepared from the extracted RNA byusing an RNA reverse transcriptase (AccuPower CycleScript RTPremix/dT₂₀, BIONEER, Korea), as follows.

Specifically, 1 μg of the extracted RNA was put in each of 0.25 mlEppendorf tubes containing AccuPower CycleScript RT Premix/dT20(BIONEER, Korea), and the distilled water treated with diethylpyrocarbonate (DEPC) was added thereto to have a total volume of 20 μl.By using a PCR machine (MyGenie™ 96 Gradient Thermal Block, BIONEER,Korea), two steps of RNA-primer hybridization at 30° C. for 1 minute andpreparation of cDNA at 52° C. for 4 minutes were repeated six times.Then inactivation of enzyme was performed at 95° C. for 5 minutes tofinish the amplification reaction.

Example 6-3-2 Relative Quantitative Analysis of Survivin Gene mRNA

The relative quantity of survivin gene mRNA was quantitated through therealtime PCR by using the cDNA prepared in the example 6-3-1 as atemplate as follows.

That is to say, the cDNA prepared in the example 6-3-1 was ⅕-diluted ineach well of a 96-well plate, and then 3 μl of the diluted cDNA, 10 μlof 2× GreenStar™ PCR master mix (BIONEER, Korea), 6 μl of distilledwater, and 1 μl of survivin qPCR primer (10 pmole/μl each, BIONEER,Korea) were used to prepare a mixture liquid in order to analyze thesurvivin expression level. On the other hand, by using HMBS(Hydroxymethyl-bilane synthase), HPRT1 (Hypoxanthinephosphoribosyl-transferase1), UBC (Ubiquitin C), YWHAZ (Tyrosine3-monooxygenase/tryptophan 5-monooxygenase activation protein, zetapolypeptide), which are housekeeping genes (hereafter, referred to as“HK genes”), as the reference gene, in order to normalize the mRNAexpression level, the cDNA prepared in the example 6-3-1 was ⅕-diluted,and then 3 μl of the diluted cDNA, 10 μl of 2× GreenStar™ PCR master mix(BIONEER, Korea), 6 μl of distilled water, and 1 μl of qPCR primer ofeach HK gene (10 pmole/μl each, BIONEER, Korea) were input to prepare aHK gene realtime PCR mixture liquid in each well of the 96-well plate.The following reaction was performed on the 96-well plate containing themixture liquid by using an Exicycler™ 96 Real-Time Quantitative ThermalBlock (BIONEER, Korea).

Enzyme activation and secondary structure of cDNA were removed by thereaction at 95° C. for 15 minutes. Then, four steps of denaturing at 94°C. for 30 seconds, annealing at 58° C. for 30 seconds, extension at 72°C. for 30 seconds, and SYBR green scan were repetitively performed 42times, and then the final extension at 72° C. for 3 minutes wasperformed. Then the temperature was kept at 55° C. for 1 minute, and amelting curve of 55° C.˜95° C. was analyzed. After finishing the PCR,the survivin Ct (threshold cycle) values obtained respectively werecorrected by using the mRNA values (normalization factor, NF) normalizedthrough the HK genes, and then Δvalue was obtained between the Ct valueof a control group treated with only the transfection reagent and thecorrected Ct values. The expression rates of survivin mRNA were comparedwith one another by using the ΔCt values and the calculation equation of2^((−ΔCt))×100 (FIG. 19).

As a result, as shown in FIG. 19, the siRNA PEG conjugates of theconjugates 2 to 5 highly inhibit the survivin mRNA level, compared withthe siRNA-polymer compound conjugate 1, unlike the result of the case inwhich transfection was performed through the transfection reagent. ThesiRNA-polymer compound conjugates 1 to 5 exhibited higher RNAi effect ina low concentration (500 nM) than in a high concentration. In addition,the siRNA-hydrophobic polymer compound conjugates of the conjugates 9 to14 exhibited a lower inhibition of the survivin mRNA expression level,compared with the siRNA conjugates 1 to 5, when treated at the sameconcentration (500 nM). However, when treated at the high concentrationcondition (1 uM), particularly the end modification of the siRNA-polymercompound conjugate 14 leads to high inhibition effect of the survivinmRNA expression level.

SEQUENCE LISTING

See sequence listing

1. A nanoparticle consisting of siRNA conjugates, wherein the siRNA conjugate has following structure: A-X-R-Y-B wherein one of A and B is a hydrophilic polymer compound and the other is a hydrophobic compound; X and Y are independently a simple covalent bond or a linker-mediated covalent bond; and R is siRNA.
 2. A nanoparticle of claim 1, wherein the hydrophilic polymer compound and the hydrophobic compound in the siRNA conjugate are conjugated to the same strand of siRNA.
 3. A nanoparticle of claim 1, wherein the hydrophilic polymer compound and the hydrophobic compound in the siRNA conjugate are conjugated to both strands of siRNA.
 4. A nanoparticle of claim 1, wherein a single strand of the siRNA in the siRNA conjugate comprises 19 to 31 nucleotides.
 5. A nanoparticle of claim 1, wherein the hydrophobic compound in the siRNA conjugate has a molecular weight of 250 to 1,000.
 6. A nanoparticle of claim 1, wherein the hydrophobic compound in the siRNA conjugate is C₁₆˜C₅₀ hydrocarbon or cholesterol.
 7. A nanoparticle of claim 1, wherein the covalent bond (X, Y) in the siRNA conjugate is a non-degradable bond or a degradable bond.
 8. A nanoparticle of claim 7, wherein the non-degradable bond is an amide bond or a phosphate bond.
 9. A nanoparticle of claim 7, wherein the degradable bond is selected from a disulfide bond, an acid-cleavable bond, an ester bond, an anhydride bond, a biodegradable bond and an enzyme-cleavable bond.
 10. A nanoparticle of claim 1, wherein the hydrophilic polymer compound in the siRNA conjugate is a non-ionic polymer compound having a molecular weight of 1,000 to 10,000.
 11. A nanoparticle of claim 1, wherein the hydrophilic polymer compound in the siRNA conjugate is selected from a group consisting of polyethylene glycol (PEG), polyvinylpyrolidone, and polyoxazoline.
 12. A pharmaceutical composition comprising a pharmaceutically effective amount of the nanoparticles of claim
 1. 